Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011/01/13 -2012/11/27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to EU guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
- Sex: 3 Males and 3 Females. The females assigned to test were nulliparous and non-pregnant.
- Age/Body weight: Young adult (10-11 weeks)/males 288-309 grams and females 196-209 grams at experimental start.
- Housing: The animals were singly housed in suspended stainless steel caging with mesh floors, which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Litter paper was placed beneath the cage and was changed at least three times per week.
- Animal Room Temperature and Relative Humidity Ranges: 19-23ºC and 47-62%, respectively.
- Animal Room Air Changes/Hour: 12. Airflow measurements are evaluated regularly and the records are kept on file at Product Safety Labs.
- Photoperiod: 12-hour light/dark cycle (light period 0500-1700 hours)
- Acclimation Period: 19 days
- Food: Harlan Teklad Global 16% Protein Rodent Diet® #2016. The diet was available ad libitum, except during the exposure.
- Water: Filtered tap water was supplied ad libitum by an automatic water dispensing system except during exposure.
- Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at Product Safety Labs.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
A. Pre-Test Trials
Prior to initiation of the full inhalation study, pre-test trials were conducted to establish generation procedures to achieve, to the extent possible, the desired chamber concentration (5.0 mg/L) and desired particle size distribution (mass median aerodynamic diameter between 1 and 4 μm). In these trials, the following adjustments were made in an attempt to achieve these objectives:
Air Pressure: constant
Compressed Generator Airflow: constant
Compressed Mixing Airflow: constant
Total Airflow: constant
Motor Setting: varied
Dust Generating System: constant
Cutting Head: constant
Cutting Blade: constant
Packing Pressure: constant
Material Preparation: constant
The procedures and aerosolization equipment used in the full test were based on the results of pre-test trial number 3. This provided a chamber concentration of 5.13 mg/L and a mass median aerodynamic diameter of 2.88 μm.

B. Inhalation Procedures
The exposure chamber, air supply and equipment used to measure particle size distribution, airflow and chamber concentration were the same as used during the pre-test trials and are described below.

Nose-Only Exposure Chamber: A nose-only inhalation chamber with an internal volume of approximately 6.7 liters (Nose Only Inhalation Chamber, ADG Developments LTD) was used for exposure. Animals were individually housed in polycarbonate holding tubes which seal to the chamber with an “O” ring during exposure. The base unit terminates the chamber with a 0.5-inch diameter tube for discharged air.

Air Supply: Approximately 30.0 liters per minute (Lpm) of filtered air and an additional 6.0 Lpm of compressed mixing air was supplied by an air compressor (Powerex Model: SES05) to the dust generator to help uniformly distribute the test atmosphere by creating a vortex at the chamber inlet. Compressed airflow was measured with a Mass Flowmeter. Chamber airflow was monitored throughout the exposure period and recorded periodically. Total airflow was 36.0 Lpm. Based on the volume of the inhalation chamber, this airflow provided approximately 322 air changes per hour during the study. The exposure was conducted under slightly negative pressure.

Ambient Conditions: The exposure tube temperature and relative humidity ranges during exposure were 21-23ºC and 12-19%, respectively. The room temperature and relative humidity ranges during exposure were 21-23ºC and 37-44%, respectively. In-chamber measurements and room conditions were measured with a Temperature-Humidity Monitor (Fisher Scientific, Model #11-661-18). Temperature and relative humidity values were recorded every 15 minutes for the first hour of exposure and every 30 minutes thereafter.

Dust Generation: The test substance was aerosolized using a modified Wright Dust Generator driven by a variable speed motor D.C. speed control with 0-100 potentiometer. The test substance was packed into the dust container and compressed to 2,000 lbs/in2 using a lab press. The container was then fitted with a stainless steel cutting head and cutting blade. Compressed/mixing air was supplied to the dust generator at 30 psi. The aerosolized dust was then fed directly into the chamber through the dust outlet assembly.

Chamber Concentration Measurements: Gravimetric samples were withdrawn at six intervals from the breathing zone of the animals. Samples were collected using mm glass fiber filters in a filter holder attachedto a vacuum pump. Filter papers were weighed before and after collection to determine the mass collected. This value was divided by the total volume of air sampled to determine the chamber concentration. The collections were carried out for 1 minute at airflows of 4 Lpm. Sample airflows were measured using a Mass Flowmeter.

Particle Size Distribution: An eight-stage ACFM Anderson Ambient Particle Sizing Sampler was used to assess the particle size distribution of the test atmosphere. Samples were withdrawn from the breathing zone of the animals at two intervals. The filter paper collection stages were weighed before and after sampling to determine the mass collected upon each stage. The aerodynamic mass median diameter and geometric standard deviation were determined graphically using two-cycle logarithmic probit axes.

Exposure Period: The animals were exposed to the test atmosphere for 4 hours. The exposure period was extended beyond 4 hours to allow the chamber to reach equilibrium. The times for 90 and 99% equilibration of the chamber atmosphere were 0.4 and 0.9 minutes, respectively. At the end of the exposure period, the generation was terminated and the chamber was operated for a further 24 minutes with clean air. At the end of this period the animals were removed from the exposure tube. Prior to being returned to their cages, excess test substance was removed from the fur of each animal.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
ca. 4 h
Remarks on duration:
one additional minute was necessary to ensure atmosphere equilibration
Concentrations:
5.09 mg/L
No. of animals per sex per dose:
3 animals per sex and per dose
Control animals:
no
Details on study design:
Selection of animals:
On the day of and prior to exposure, the rats were examined for health and weighed. Six healthy, naive rats (three males and three females; not previously tested) were selected for test.

Body Weights
Individual body weights of the animals were recorded prior to test substance exposure (initial) and on Days 1, 3, 7 and 14 (termination) following exposure.


Cage-side observation:
All animals were observed for mortality during the exposure period. The animals were examined for signs of gross toxicity, and behavioral changes upon removal from the exposure tube and at least once daily thereafter for 14 days. Observations included gross evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, and coma.

Necropsy:
All rats were euthanized via CO2 inhalation on Day 14. Gross necropsies were performed on all animals. Tissues and organs of the thoracic and abdominal cavities were examined.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.09 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality observed
Clinical signs:
other: Following exposure, all animals exhibited irregular respiration. However, all animals recovered from this symptom by Day 2 and appeared active and healthy for the remainder of the 14-day observation period.
Body weight:
Although all animals lost body weight by Day 1, all animals showed a continued weight gain thereafter through Day 14.
Gross pathology:
No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.

Any other information on results incl. tables

TABLE 2: PRE-TEST EXPOSURE TRIALS

 Trial N°  Compressed/Mixing Air Pressure (psi)    Compressed Air Volume (Lpm)  Compressed Mixing Air (Lpm) Total Air Volume (Lpm)   Dust Generator Motor Setting  Packing Pressure (lbs/in2)  Chamber Conc. (mg/L)  Particle Size Sampled
 1 30/30   30.0  6.0  36.0  15.0  2,000  11.60  No
 2  30/30  30.0  6.0  36.0  10.0  2,000  6.88  No
 3 30/30  30.0   6.0  36.0  8.0  2,000  5.13  Yes

TABLE 3: SUMMARY OF PRE-TEST EXPOSURE TRIAL

 Trial No.  Chamber Concentration (mg/L)  Mass Median Aerodynamic Diameter2 (μm)
 3  5.13  2.88

TABLE 4: GRAVIMETRIC CHAMBER CONCENTRATIONS

Sample Number Chamber Time of Sample (hour)   Mass Collected (mg) Airflow Sampled (Lpm)  Collection Time (min)   Concentration (mg/L) 
 1  0.5  20.4  4  1  5.10
 2  1  20.3  4  1  5.08
 3  2  21.2  4  1  5.30
 4  2.5  20.3  4  1  5.08
 5  3.5  19.5  4  1  4.88
 6  3.75  20.4  4  1  5.10

Average + standrad deviation: 5.09 + 0.13

Table 4: SUMMARY OF PARTICULE SIZE DISTRIBUTION:

 Sample No. Time of Sample (hours)   Collection Time (minutes) Mass Median Aerodynamic Diameter (μm)   Geometric Standard Deviation
 1  1.5  3.07  2.17
 2  3 2.86  2.14 

These two samples show a medium MMAD of 2.97 µm

TABLE 7: INDIVIDUAL BODY WEIGHTS

 Animal No.  Sex  Body Weight (g)  Body Weight (g)   Body Weight (g)  Body Weight (g)   Body Weight (g) 
     Initial  Day 1 Day 3  Day 7  day 14 
 3301 309  296 304  322  340 
 3302 288  280  285  300  315 
 3303 305  294  300  318  330 
 3304 196  194  195  207  211 
3305  209  206  209  219  230 
 3306 200  194  199  209  217 

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the single exposure acute inhalation LC50 of the test substance is greater than 5.09 mg/L (limit test, OECD) in male and female rats.

In accordance with the provisions of Regulation (EC) No. 1272/2008 on the Classification, Labeling, and Packaging of Substances and Mixtures, classification is not required based on the results of this study.
Executive summary:

The gravimetric and nominal chamber concentrations were 5.09 mg/L and 9.31 mg/L, respectively. Based on graphic analysis of the particle size distribution as measured with an ACFM Andersen Ambient Particle Sizing Sampler, the mass median aerodynamic diameter was estimated to be 2.97 μm. The total amount of test substance used was 81.0 grams.

All animals survived exposure to the test atmosphere. Following exposure, all animals exhibited irregular respiration. However, all animals recovered from this symptom by Day 2 and appeared active and healthy for the remainder of the 14-day observation period. Although all animals lost body weight by Day 1, all animals showed a continued weight gain thereafter through Day 14. No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period. Therefore, it was concluded that the LC 50 for male and female rats was > 5.09 mg/L.